Fig 2: Respiratory syncytial virus (RSV) infection promotes phospho-myosin light chain 2 (pMLC2) expression. 16HBE14o (16HBE) cells were infected for 48 h with medium control (C), live RSV (R; 0.5 multiplicity of infection), or UV-inactivated RSV (UV-RSV; U) followed by cell detachment and seeding on collagen type I-coated plates. After 2 h, nonattached cells were washed off, and attached cells were fixed and immunolabeled with pMLC2 antibody (A). Scale bar = 5 µm. Total cell fluorescence was measured using ImageJ software (B). Western blot analysis of 16HBE control and RSV-infected cells was performed using total cell lysates. Expression of pMLC2 and total MLC2 were analyzed by Western blot, followed by densitometric analysis, normalized to total MLC2 and GAPDH, respectively (C-E). Data are presented as means ± SD; n = 3 independent experiments. Student’s two-tailed t test was used for the results shown in B (n = 4); a paired Student’s one-tailed t test was used for the results shown in D and E. *P < 0.05 and ***P < 0.001 versus the respective control as determined.

Fig 3: VEC contractility is involved in filopodia formation and encapsulation of eggs.(A) Immunostaining of phosphor-MLC2 and F-actin in VECs during encapsulation of live mature eggs at 4 h. The egg location is confirmed by different focal planes of VEC F-actin staining given that the eggshell is covered by VECs. The white arrows in the middle F-actin focal plane show the extended VECs surrounding the egg and indicate the corresponding egg location. (B) Fluorescent intensity analysis (the right panel) of phosphor-MLC2 basal (red line)/ F-actin basal (gray line) and F-actin middle (green line)/ F-actin basal (gray line) shows the distributions of phosphor-MLC2 and egg location across the merged images (yellow dashed line). Note how the induction of VEC myosin contractility localizes with the egg location (scale bar: 50 µm). (C) Phosphor-MLC2 fluorescent integrative density (mean ± s.e.m.) after 4 h of egg-VEC interaction. VECs were pre-treated with DMSO (white), Y27632 (Rho/RCOCK pathway inhibitor; gray) and ML-7 (myosin light chain kinase inhibitor; blue) for 30 min before incubating with eggs. * and ****, indicate p < 0.05 and p < 0.0001, respectively using a one-way ANOVA with Dunnett’s post-test. Six image fields from three independent experiments were analyzed. (D) Quantification of the mature egg portion covered by VECs (mean ± s.e.m.). * indicates p < 0.05 using Student’s t-test. (E) Quantification of the number (mean ± s.e.m.) of FPs and NT formed per mature egg. *** indicates p < 0.001 and ### indicates p < 0.001 using Student’s t-test. (F) Quantification of the average lengths of FPs and NTs (mean ± s.e.m.). ** indicates p < 0.01 and # indicates p<0.05 using Student’s t-test. For D–F, 16 and 11 eggs from three independent experiments were used for the control and Y-27632 treated VEC cases, respectively.

Fig 4: ROCK2 deletion does not inhibit doxorubicin-induced cell detachment. (a) Representative images of rhodamine-phalloidin staining for F-actin (red) and DAPI staining (blue) of WT and ROCK2-/- cells treated with 3 µM doxorubicin for 16 h. Actomyosin contractile rings are indicated with white arrow, and folded periphery membranes with white arrowheads. Bar, 50 µm. (b) Cells showing formation of cortical contractile ring structure or with folded periphery membrane were counted and expressed as percentage of total cells under indicated conditions. For each treatment, over 500 cells were counted. (c) Floating cells and attached cells were separately collected and counted at indicated time points and dosages of doxorubicin. Floating cell ratio was expressed as percentage of total cells (floating plus attached cells) under indicated conditions. (d and e) Representative image (top) and quantitative analysis (bottom) of western blot of p-MLC2 and MLC2, or p-cofilin and cofilin in cell lysates from attached WT and ROCK2-/- MEFs treated with 3 µM doxorubicin for 30 min. *P<0.05 versus control of the same genotype. #P<0.05 versus WT under the same treatment condition

Fig 5: Myosin-II dynamics in nocodazole-treated cells. a Upregulation of phosphorylated MLC2 (p-MLC2) after nocodazole treatment, assessed by western blotting. Densitometric comparison for the electrophoretic bands is also shown. n = 4 independent experiments, mean ± SEM. b Immunostaining for p-MLC2, F-actin, and E-cadherin in HT29 cells. c Lateral views of HT29 cells immunostained as in b. d Densitometric scans of immunosignals for p-MLC2 and E-cadherin along the yellow line drawn in b. e Time-lapse images of MLC2-EGFP expressed in HT29 cells during nocodazole treatment. Images at the right are a close-up view of the cell indicated by arrow at the left panel. Images are identical to Supplementary Movie 3. f Effects of aE-catenin knockdown on MHC-IIA distribution, as assessed by immunostaining of HT29 cells transfected with control or aE-catenin specific siRNA. Images were obtained with Airyscan. g Effects of nocodazole treatment on the distribution of p-MLC2 and E-cadherin in Colo205, LS180, and SW620 cells. Scale bars, 10 µm b, c, f left and g; 2 µm (f right). See also Supplementary Fig. 5